Our goal is to understand the potential independent and combined contributions of several critical pathways for human development to risks of orofacial clefts. These pathways include methyl group metabolism, oxidative stress, glycemic control, and the hypothalamic-pituitary adrenocortical (HPA) axis. Each of these pathways has evidence for its contribution to clefts. Here we propose two sets of analyses that will substantially extend our knowledge regarding the contribution of these pathways to clefts. First, we will explore nutrient exposures that regulate these pathways. Available evidence supports an association of nutritional status with clefts. Most studies have investigated one or two nutrients at a time. We propose to extend current knowledge by examining multiple nutrients simultaneously, and using an innovative pathway-focused analytic approach, as reflected in Aim 1 - Nutrient pathways and clefts: We will examine the association of risk of clefts with nutritional exposures that contribute to several specific mechanistic pathways, using data from a population- based case-control study we conducted in California. The pathways and exposures are as follows: 1) oxidative stress - nutrients that protect against oxidative stress (1- and 2-carotene, cryptoxanthin, cysteine, folate, lutein, lycopene, oleic acid, pro-vitamin A, vitamins C and E, zinc) or promote it (iron, saturated fat);2) methyl group metabolism - betaine, choline, folate, methionine, riboflavin, vitamin B6, vitamin B12, zinc;and 3) glycemic control - dietary glycemic load;intake of glucose, sucrose, fructose, fiber. For our second Aim, we will consider the HPA axis pathway. The HPA axis represents a complex feedback system by which the body responds to stress. Experimental studies indicate that corticosteroids, a product of the stress response, cause clefts. Several studies in humans have examined clefts and stress, and most found an increased risk associated with higher stress. Most of these studies have been quite limited in their assessment of stress. None of them have included measures of social support, an important potential buffer of the negative effects of stress. To further extend our understanding of stress and clefts, we propose the following specific aim: Aim 2 - Stress and clefts: We will examine the association of risk of clefts with maternal stressful life events and social support, using data from the National Birth Defects Prevention Study (NBDPS), a population-based case-control study. The proposed research will move forward our understanding of the causes of clefts and is innovative and unique, the research team is highly experienced and successful in conducting epidemiologic research on birth defects, and the data to be used are population-based, rigorously collected, and high quality. PUBLIC HEALTH RELEVANCE: The proposed research will improve our understanding of the causes of orofacial clefts. Its findings will contribute to the development of effective interventions or prevention messages, so that this common, costly outcome can be prevented.